| Budget Amount *help |
¥12,300,000 (Direct Cost: ¥12,300,000)
Fiscal Year 2004: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥8,800,000 (Direct Cost: ¥8,800,000)
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| Research Abstract |
Our Group has been studying about structure and function of the centromere region of human chromosomes in the viewpoint of DNA/protein complex formed by interaction between DNA and proteins. We have elaborated to purify a centromere specific histone H3 variant, CENP-A, and established the method for reconstruction of the nucleosome containing CENP-A instead of histone H3 (CENP-A nucleosome)(ref.6). We have produced highly specific anti-CENP-A monoclonal antibody and this antibody has been supplied to lots of investigators inside and/or outside of this country (ref.1,11-13). We have shown that this antibody was excellent in high specificity not only for immuno-stain of the cells but also for chromatin immuno-precipitation (ref.4). We have developed the method to mildly solubilize the chromatin and isolate the centromere chromatin from interphase nuclei using anti-CENP-A antibody (Native Chromatin Immuno-Precipitation;N-ChIP)(ref.9). The isolated chromatin complex contained all the reported centromere structural proteins, CENP-A,CENP-B,CENP-C,CENP-H,CENP-l/hMis6 and hMis12. Therefore, we call this complex as Interphase CENtromere complex(I-CEN complex). The components of the I-CEN complex have been comprehensively subjected to LC/MS/MS analyses and 40 proteins were detected (ref.9). In collaboration with Dr.Naohito Nozaki, Kanagawa Dental College, we tried to produce the antibodies against 20 proteins using chemically synthesized amino-and/or carboxyl-terminal peptides as antigens. We've got 10 kinds of antibodies and 5 of them are now under analysis ; antibodies against MgcRacGAP,hMKLP-1,ASR2A,Rsf1(XAP8) and SNF2H. To analyze the physiological function of each protein we are now trying RNA-interference-mediated depletion of the proteins. By siRNA transfection we reveal that ASR2A is essential for interaction between kinetochore and microtubule (paper in preparation).
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